Small scale production

Before trying to grow new cells in a reactor, the strain had to be tested with vial-scale cultivations. In this way we could gather information about the induction, cell growth rates in TB-media, and some estimates how much time are needed for propane formation after adding IPTG. At the same time we could practise the usage of gas gromatography/mass spectrometry and make proper standards for propane. Basically, terrific broth (TB) containing induced bacteria would be added into 22ml GC-vials. There the cells are incubated certain time and propane accumulates into the head-space of the vial where the injection and analysis would happen. The protocol contains more detailed information for the preparation of samples.

Two samples were incubated 17 h with 50 RPM shaking in 22 ml vials. Propane concentrations were 111 μg/l and 88 μg/l where the litres were as in the volume of cultivation media. Furthermore, strain's growth rate was significant as the OD600 (1 ml cuvettes) increased from 0.1425 to 0.5212 for 1h 10 min time scale. The rate was assumed to be much lower because TB-media contained four different antibiotics which were needed to keep plasmids inside of bacteria. Details about standards, peaks etc. for GS/MS can be seen in this file.

Continuous production

0.5L chemostat experiment with 44 ml/h flow rate was successful. With 1.0 l/h aeration, the propane content of reactor's gas phase was determined to be 4.5-22.7 μg/l with GC/MS. The results were calculated when 0.1-0.5 μg of propane was gathered from reactor's gas exhaust into a 22ml gas chromatography vial. However, these values were below GC-standard line so the accuracy may have decreased. The GC-samples were also gathered after the second steady state was over. Nevertheless, the result proves that propane can be formed industrially with continuous production.

The batch phase took six hours, and soon after starting the process dissolved oxygen amount decreased into zero. Therefore, oxygen became the limiting factor of growth. Continuous phase was started and the first steady state was reached 39 h after starting to feed fresh media into the reactor. Overall, the steady state lasted for 15 hours. The density of cell population was calculated to be 9.85 g/L at this point, which can be assumed relatively high. Interestingly, bacteria acted as facultative anaerobe when changing its oxygen uptake depending on the growth phase.

IPTG induction started with 2mM concentration and the population growth started to react to changed process conditions. New steady state was achieved 40 h later and the population density became 9.28 g/L. Thus, there was a 0.57 g/L difference of the biomass between the normal growth and the growth with propane production. Reactor's glucose consentration decreased from original 20 g/l to zero during the exponential grow phase as the cells used it as a carbon source. However, it was hard to recognize which was the limiting factor of growth and production, glucose or oxygen? During the first steady state, glucose concentration increased when the need for the catabolism of biomass was lower.

Before starting to feed fresh media, the end of batch phase was determined from decreased pH-level when the cells produced acids as a response for nutrient deficiency. TB-media used for cultivation contained phosphates which had buffer capacity but it wasn't enough. pH-control was assembled in order to maintain pH-values above 5.0.

More information about the experiment available at the Process data sheet and the Lab book. Details about standards, peaks etc. for GS/MS can be seen in this file.